The long-time orphan protist Meringosphaera mediterranea Lohmann, 1902 [1903] is a centrohelid heliozoan

Meringosphaera is an enigmatic marine protist without clear phylogenetic affiliation, but it has long been suggested to be a chrysophyte-related autotroph. Microscopy-based reports indicate that it has a worldwide distribution, but no sequence data exist so far. We obtained the first 18S rDNA sequence for M. mediterranea (identified using light and electron microscopy) from the west coast of Sweden. Observations of living cells revealed granulated axopodia and up to 6 globular photosynthesizing bodies about 2 μm in diameter, the nature of which requires further investigation. The ultrastructure of barbed undulating spine scales and patternless plate scales with a central thickening is in agreement with previous reports. Molecular phylogenetic analysis placed M. mediterranea inside the NC5 environmental clade of Centroplasthelida (Haptista) along with additional environmental sequences, together closely related to Choanocystidae. This placement is supported by similar scales in Meringosphaera and Choanocystidae. We searched the Tara Oceans 18S V9 metabarcoding dataset, which revealed four OTUs with 94.8%–98.2% similarity, with oceanic distribution similar to that based on morphological observations. The current taxonomic position and species composition of the genus are discussed. The planktonic lifestyle of M. mediterranea contradicts the view of some authors that centrohelids enter the plankton only temporarily.     

The hierarchical packing of euchromatin domains can be described as multiplicative cascades

The genome is packed into the cell nucleus in the form of chromatin. Biochemical approaches have revealed that chromatin is packed within domains, which group into larger domains, and so forth. Such hierarchical packing is equally visible in super-resolution microscopy images of large-scale chromatin organization. While previous work has suggested that chromatin is partitioned into distinct domains via microphase separation, it is unclear how these domains organize into this hierarchical packing. A particular challenge is to find an image analysis approach that fully incorporates such hierarchical packing, so that hypothetical governing mechanisms of euchromatin packing can be compared against the results of such an analysis. Here, we obtain 3D STED super-resolution images from pluripotent zebrafish embryos labeled with improved DNA fluorescence stains, and demonstrate how the hierarchical packing of euchromatin in these images can be described as multiplicative cascades. Multiplicative cascades are an established theoretical concept to describe the placement of ever-smaller structures within bigger structures. Importantly, these cascades can generate artificial image data by applying a single rule again and again, and can be fully specified using only four parameters. Here, we show how the typical patterns of euchromatin organization are reflected in the values of these four parameters. Specifically, we can pinpoint the values required to mimic a microphase-separated state of euchromatin. We suggest that the concept of multiplicative cascades can also be applied to images of other types of chromatin. Here, cascade parameters could serve as test quantities to assess whether microphase separation or other theoretical models accurately reproduce the hierarchical packing of chromatin.     

Different metastasis promotive potency of small G-proteins RalA and RalB in in vivo hamster tumor model

Background

Previously we have shown that oncogenic Ha-Ras stimulated in vivo metastasis through RalGEF-Ral signaling. RalA and RalB are highly homologous small G proteins belonging to Ras superfamily. They can be activated by Ras-RalGEF signaling pathway and influence cellular growth and survival, motility, vesicular transport and tumor progression in humans and in animal models. Here we first time compared the influence of RalA and RalB on tumorigenic, invasive and metastatic properties of RSV transformed hamster fibroblasts.

Methods

Retroviral vectors encoding activated forms or effector mutants of RalA or RalB proteins were introduced into the low metastatic HET-SR cell line. Tumor growth and spontaneous metastatic activity (SMA) were evaluated on immunocompetent hamsters after subcutaneous injection of cells. The biological properties of cells, including proliferation, clonogenicity, migration and invasion were determined using MTT, wound healing, colony formation and Boyden chamber assays respectively. Protein expression and phosphorylation was detected by Westen blot analysis. Extracellular proteinases activity was assessed by substrate-specific zymography.

Results

We have showed that although both Ral proteins stimulated SMA, RalB was more effective in metastasis stimulation in vivo as well as in potentiating of directed movement and invasion in vitro. Simultaneous expression of active RalA and RalB didn't give synergetic effect on metastasis formation. RalB activity decreased expression of Caveolin-1, while active RalA stimulated MMP-1 and uPA proteolytic activity, as well as CD24 expression. Both Ral proteins were capable of Cyclin D1 upregulation, JNK1 kinase activation, and stimulation of colony growth and motility. Among three main RalB effectors (RalBP1, exocyst complex and PLD1), PLD1 was essential for RalB-dependent metastasis stimulation.

Conclusions

Presented results are the first data on direct comparison of RalA and RalB impact as well as of RalA/RalB simultaneous expression influence on in vivo cell metastatic activity. We showed that RalB activation significantly more than RalA stimulates SMA. This property correlates with the ability of RalB to stimulate in vitro invasion and serum directed cell movement. We also found that RalB-PLD1 interaction is necessary for the acquisition of RalB-dependent high metastatic cell phenotype. These findings contribute to the identification of molecular mechanisms of metastasis and tumor progression.     

The structural peculiarities of condensed DNA micro- and nanoparticles formed in PCR

Studies of DNA condensation have opened new perspectives in biotechnology and medicine. DNA condensation induced by polyamines or trivalent metal ions in vitro at room temperature has been investigated in detail. Our recent studies have demonstrated Mg²⁺-mediated formation of DNA condensates during the PCR. In this study, we report the unique morphology and fine structure of PCR-generated condensed DNA particles using electron and atomic force microscopy. The principal morphologies of studied DNA condensates are 3D particles of micrometer dimensions, oval microdisks of nanometer thickness, filaments, and compact nano-sized particles. SEM examinations have revealed a new structural type of spherical and elliptical 3D microparticles formed by numerous definitely oriented microdisks and their segments. AFM revealed a granular structure of the microdisk surface and the smallest nano-sized disks and thinnest nanofibrils – that appear to be the primary products of DNA condensation during the PCR. We suggest that the formation of DNA nanofibrils and nanodisks in PCR occurs due to Mg²⁺ – mediated intermolecular (lateral) and intramolecular condensation of ssDNA. Aggregation of elementary nanodisks in the course of thermal PCR cycles, occurring both by magnesium cations and via complementary interactions, give a rise to large nano-sized aggregates and more complex microparticles.     

Regulation of electron transport in C(3) plant chloroplasts in situ and in silico: short-term effects of atmospheric CO(2) and O(2)

In this work, we have investigated the effects of atmospheric CO(2) and O(2) on induction events in Hibiscus rosa-sinensis leaves. These effects manifest themselves as multiphase kinetics of P(700) redox transitions and non-monotonous changes in chlorophyll fluorescence. Depletion of CO(2) and O(2) in air causes a decrease in linear electron flux (LEF) and dramatic lowering of P(700)(+) level. This is explained by the impediment to electron efflux from photosystem 1 (PS1) at low acceptor capacity. With the release of the acceptor deficit, the rate of LEF significantly increases. We have found that oxygen promotes the outflow of electrons from PS1, providing the rise of P(700)(+) level. The effect of oxygen as an alternative electron acceptor becomes apparent at low and ambient concentrations of atmospheric CO(2) < or = 0.06-0.07%). A decrease in LEF at low CO(2) is accompanied by a significant (about 3-fold) rise of non-photochemical quenching (NPQ) of chlorophyll fluorescence. Such an increase in NPQ can be explained by more significant acidification of the thylakoid lumen. This occurs due to lessening the proton flux through the ATP synthases caused by a decrease in the ATP consumption in the Bassham-Benson-Calvin (BBC) cycle. pH-dependent mechanisms of electron transport control have been described within the frames of our mathematical model. The model describes the reciprocal changes in LEF and NPQ and predicts the redistribution of electron fluxes on the acceptor side of PS1. In particular, the contribution of cyclic electron flow around PS1 (CEF1) and water-water cycle gradually decays during the induction phase. This result is consistent with experimental data indicating that under the steady-state conditions the contribution of CEF1 to photosynthetic electron transport in Hibiscus rosa-sinensis is insignificant (< or = 10%).     

Synthesis of 1,2-cis- and 1,2-trans-glycosides of 2-acetamido-4,6-O-benzylidene-2-deoxy-D-glucopyranose by anomeric O-alkylation

The reaction of a partially protected 1-hydroxy derivative of N-acetyl-D-glucosamine with benzyl bromide under conditions of anomeric O-alkylation was studied. It was found that the stereoselectivity of the reaction depended on the nature of the alkali metal cation constituent of a transient ion pair. The substitution of the Li(+) cation for K(+) or complexation with a crown ether allowed the steric outcome to be shifted from β- to α-selectivity.     

Nodal cis-regulatory elements reveal epiblast and primitive endoderm heterogeneity in the peri-implantation mouse embryo

Nodal, a secreted factor known for its conserved functions in cell-fate specification and the establishment of embryonic axes, is also required in mammals to maintain the pluripotency of the epiblast, the tissue that gives rise to all fetal lineages. Although Nodal is expressed as early as E3.5 in the mouse embryo, its regulation and functions at pre- and peri-implantation stages are currently unknown. Sensitive reporter transgenes for two Nodal cis-regulatory regions, the PEE and the ASE, exhibit specific expression profiles before implantation. Mutant and inhibitor studies find them respectively regulated by Wnt/β-catenin signaling and Activin/Nodal signaling, and provide evidence for localized and heterogeneous activities of these pathways in the inner cell mass, the epiblast and the primitive endoderm. These studies also show that Nodal and its prime effector, FoxH1, are not essential to preimplantation Activin/Nodal signaling. Finally, a strong upregulation of the ASE reporter in implanting blastocysts correlates with a downregulation of the pluripotency factor Nanog in the maturing epiblast. This study uncovers conservation in the mouse blastocyst of Wnt/β-catenin and Activin/Nodal-dependent activities known to govern Nodal expression and the establishment of polarity in the blastula of other deuterostomes. Our results indicate that these pathways act early on to initiate distinct cell-specification processes in the ICM derivatives. Our data also suggest that the activity of the Activin/Nodal pathway is dampened by interactions with the molecular machinery of pluripotency until just before implantation, possibly delaying cell-fate decisions in the mouse embryo.